Isolated human enzyme proteins, nucleic acid molecules encoding human enzyme proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the enzyme peptides, and methods of identifying modulators of the enzyme peptides.

SUMMARY OF THE INVENTION

[0001] The present invention is based in part on the identification ofamino acid sequences of human enzyme peptides and proteins that arerelated to the trypsin-like serine protease subfamily, as well asallelic variants and other mammalian orthologs thereof. These uniquepeptide sequences, and nucleic acid sequences that encode thesepeptides, can be used as models for the development of human therapeutictargets, aid in the identification of therapeutic proteins, and serve astargets for the development of human therapeutic agents that modulateenzyme activity in cells and tissues that express the enzyme.

DESCRIPTION OF THE FIGURE SHEETS

[0002]FIG. 1 provides the nucleotide sequence of a transcript sequencethat encodes the enzyme protein of the present invention. (SEQ ID NO:1)In addition, structure and functional information is provided, such asATG start, stop and tissue distribution, where available, that allowsone to readily determine specific uses of inventions based on thismolecular sequence.

[0003]FIG. 2 provides the predicted amino acid sequence of the enzyme ofthe present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0004]FIG. 3 provides genomic sequences that span the gene encoding theenzyme protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at ninedifferent nucleotide positions, including two non-synonymous codingSNPs.

DETAILED DESCRIPTION OF THE INVENTION General Description

[0005] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a enzyme protein or part of aenzyme protein and are related to the trypsin-like serine proteasesubfamily. Utilizing these sequences, additional genomic sequences wereassembled and transcript and/or cDNA sequences were isolated andcharacterized. Based on this analysis, the present invention providesamino acid sequences of human enzyme peptides and proteins that arerelated to the trypsin-like serine protease subfamily, nucleic acidsequences in the form of transcript sequences, cDNA sequences and/orgenomic sequences that encode these enzyme peptides and proteins,nucleic acid variation (allelic information), tissue distribution ofexpression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to theenzyme of the present invention.

[0006] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known enzyme proteins of thetrypsin-like serine protease subfamily and the expression patternobserved. The art has clearly established the commercial importance ofmembers of this family of proteins and proteins that have expressionpatterns similar to that of the present gene. Some of the more specificfeatures of the peptides of the present invention, and the uses thereof,are described herein, particularly in the Background of the Inventionand in the annotation provided in the Figures, and/or are known withinthe art for each of the known trypsin-like serine protease family orsubfamily of enzyme proteins.

Specific Embodiments Peptide Molecules

[0007] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of theenzyme family of proteins and are related to the trypsin-like serineprotease subfamily (protein sequences are provided in FIG. 2,transcript/cDNA sequences are provided in FIG. 1 and genomic sequencesare provided in FIG. 3). The peptide sequences provided in FIG. 2, aswell as the obvious variants described herein, particularly allelicvariants as identified herein and using the information in FIG. 3, willbe referred herein as the enzyme peptides of the present invention,enzyme peptides, or peptides/proteins of the present invention.

[0008] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the enzyme peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0009] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0010] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0011] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theenzyme peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0012] The isolated enzyme peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. For example, a nucleic acid molecule encoding the enzymepeptide is cloned into an expression vector, the expression vectorintroduced into a host cell and the protein expressed in the host cell.The protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques. Manyof these techniques are described in detail below.

[0013] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). The amino acid sequence of such a protein is providedin FIG. 2. A protein consists of an amino acid sequence when the aminoacid sequence is the final amino acid sequence of the protein.

[0014] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0015] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the enzyme peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0016] The enzyme peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a enzyme peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the enzyme peptide. “Operatively linked”indicates that the enzyme peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the enzyme peptide.

[0017] In some uses, the fusion protein does not affect the activity ofthe enzyme peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant enzyme peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0018] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al, Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A enzyme peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the enzyme peptide.

[0019] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0020] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the enzyme peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0021] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0022] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0023] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0024] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the enzyme peptides of the present invention as well as beingencoded by the same genetic locus as the enzyme peptide provided herein.The gene encoding the novel enzyme of the present invention is locatedon a genome component that has been mapped to human chromosome 5 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data.

[0025] Allelic variants of a enzyme peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the enzyme peptide as well asbeing encoded by the same genetic locus as the enzyme peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. The gene encoding the novel enzyme of the presentinvention is located on a genome component that has been mapped to humanchromosome 5 (as indicated in FIG. 3), which is supported by multiplelines of evidence, such as STS and BAC map data. As used herein, twoproteins (or a region of the proteins) have significant homology whenthe amino acid sequences are typically at least about 70-80%, 80-90%,and more typically at least about 90-95% or more homologous. Asignificantly homologous amino acid sequence, according to the presentinvention, will be encoded by a nucleic acid sequence that willhybridize to a enzyme peptide encoding nucleic acid molecule understringent conditions as more fully described below.

[0026]FIG. 3 provides information on SNPs that have been found in thegene encoding the enzyme of the present invention. SNPs were identifiedat 9 different nucleotide positions, including non-synonymous codingSNPs at positions 2263 and 13195 (protein positions 31 and 164,respectively). Changes in the amino acid sequence caused by these SNPsis indicated in FIG. 3 and can readily be determined using the universalgenetic code and the protein sequence provided in FIG. 2 as a reference.

[0027] Paralogs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide, as being encoded by a gene from humans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a enzymepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0028] Orthologs of a enzyme peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the enzyme peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a enzyme peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0029] Non-naturally occurring variants of the enzyme peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the enzyme peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a enzyme peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0030] Variant enzyme peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0031] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0032] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as enzyme activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0033] The present invention further provides fragments of the enzymepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0034] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a enzyme peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the enzyme peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe enzyme peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0035] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inenzyme peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0036] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0037] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

[0038] Accordingly, the enzyme peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature enzyme peptide is fused withanother compound, such as a compound to increase the half-life of theenzyme peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature enzyme peptide, such as aleader or secretory sequence or a sequence for purification of themature enzyme peptide or a pro-protein sequence.

Protein/Peptide Uses

[0039] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a enzyme-effectorprotein interaction or enzyme-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0040] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0041] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, enzymes isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the enzyme. A large percentage ofpharmaceutical agents are being developed that modulate the activity ofenzyme proteins, particularly members of the trypsin-like serineprotease subfamily (see Background of the Invention). The structural andfunctional information provided in the Background and Figures providespecific and substantial uses for the molecules of the presentinvention, particularly in combination with the expression informationprovided in FIG. 1. Such uses can readily be determined using theinformation provided herein, that which is known in the art, and routineexperimentation.

[0042] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to enzymes that are related tomembers of the trypsin-like serine protease subfamily. Such assaysinvolve any of the known enzyme functions or activities or propertiesuseful for diagnosis and treatment of enzyme-related conditions that arespecific for the subfamily of enzymes that the one of the presentinvention belongs to, particularly in cells and tissues that express theenzyme.

[0043] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the enzyme, as a biopsyor expanded in cell culture. In an alternate embodiment, cell-basedassays involve recombinant host cells expressing the enzyme protein.

[0044] The polypeptides can be used to identify compounds that modulateenzyme activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the enzyme.Both the enzymes of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the enzyme. These compounds can befurther screened against a functional enzyme to determine the effect ofthe compound on the enzyme activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the enzyme to a desired degree.

[0045] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the enzyme protein and a molecule that normally interacts withthe enzyme protein, e.g. a substrate or a component of the signalpathway that the enzyme protein normally interacts (for example, anotherenzyme). Such assays typically include the steps of combining the enzymeprotein with a candidate compound under conditions that allow the enzymeprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the enzymeprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0046] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L- configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0047] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantenzymes or appropriate fragments containing mutations that affect enzymefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0048] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) enzyme activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate enzyme activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the enzyme proteindependent signal cascade can be assayed.

[0049] Any of the biological or biochemical functions mediated by theenzyme can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the enzyme can be assayed.

[0050] Binding and/or activating compounds can also be screened by usingchimeric enzyme proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native enzyme. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the enzyme is derived.

[0051] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the enzyme (e.g. binding partners and/or ligands).Thus, a compound is exposed to a enzyme polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble enzyme polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble enzyme polypeptide, itdecreases the amount of complex formed or activity from the enzymetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the enzyme.Thus, the soluble polypeptide that competes with the target enzymeregion is designed to contain peptide sequences corresponding to theregion of interest.

[0052] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the enzyme protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0053] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of enzyme-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a enzyme-binding protein and a candidate compound are incubated inthe enzyme protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theenzyme protein target molecule, or which are reactive with enzymeprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0054] Agents that modulate one of the enzymes of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0055] Modulators of enzyme protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the enzyme pathway, by treating cells or tissuesthat express the enzyme. These methods of treatment include the steps ofadministering a modulator of enzyme activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

[0056] In yet another aspect of the invention, the enzyme proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the enzyme and are involved in enzyme activity.Such enzyme-binding proteins are also likely to be involved in thepropagation of signals by the enzyme proteins or enzyme targets as, forexample, downstream elements of a enzyme-mediated signaling pathway.Alternatively, such enzyme-binding proteins are likely to be enzymeinhibitors.

[0057] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a enzyme proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aenzyme-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the enzyme protein.

[0058] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a enzyme-modulating agent, an antisense enzymenucleic acid molecule, a enzyme-specific antibody, or a enzyme-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0059] The enzyme proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. The method involves contacting a biologicalsample with a compound capable of interacting with the enzyme proteinsuch that the interaction can be detected. Such an assay can be providedin a single detection format or a multi-detection format such as anantibody chip array.

[0060] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0061] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered enzyme activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0062] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0063] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the enzyme protein in which one ormore of the enzyme functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and enzyme activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0064] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Accordingly, methods for treatment include the use of theenzyme protein or fragments.

Antibodies

[0065] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0066] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0067] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0068] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0069] Antibodies are preferably prepared from regions or discretefragments of the enzyme proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or enzyme/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0070] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0071] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of

[0072] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0073] The antibodies are also useful for tissue typing. Thus, where aspecific protein has been correlated with expression in a specifictissue, antibodies that are specific for this protein can be used toidentify a tissue type.

[0074] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the enzyme peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0075] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

Nucleic Acid Molecules

[0076] The present invention further provides isolated nucleic acidmolecules that encode a enzyme peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the enzyme peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0077] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5KB, 4KB,3KB, 2KB, or 1KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0078] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0079] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0080] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequencewhen the nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule.

[0081] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0082] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ IDNO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0083] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0084] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0085] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the enzyme peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0086] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0087] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the enzyme proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0088] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0089] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0090] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0091] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel enzyme of the present invention is located on a genome componentthat has been mapped to human chromosome 5 (as indicated in FIG. 3),which is supported by multiple lines of evidence, such as STS and BACmap data.

[0092]FIG. 3 provides information on SNPs that have been found in thegene encoding the enzyme of the present invention. SNPs were identifiedat 9 different nucleotide positions, including non-synonymous codingSNPs at positions 2263 and 13195 (protein positions 31 and 164,respectively). Changes in the amino acid sequence caused by these SNPsis indicated in FIG. 3 and can readily be determined using the universalgenetic code and the protein sequence provided in FIG. 2 as a reference.

[0093] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John been mapped to human chromosome 5 (as indicated in FIG.3), which is supported by multiple lines of evidence, such as STS andBAC map data.

[0094] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0095] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0096] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0097] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0098] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0099] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Accordingly, the probes can be used to detectthe presence of, or to determine levels of, a specific nucleic acidmolecule in cells, tissues, and in organisms. The nucleic acid whoselevel is determined can be DNA or RNA. Accordingly, probes correspondingto the peptides described herein can be used to assess expression and/orgene copy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease inenzyme protein expression relative to normal results.

[0100] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0101] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a enzyme protein, such as bymeasuring a level of a enzyme-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a enzymegene has been mutated.

[0102] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate enzyme nucleic acid expression.

[0103] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the enzyme gene, particularly biological and pathologicalprocesses that are mediated by the enzyme in cells and tissues thatexpress it. The method typically includes assaying the ability of thecompound to modulate the expression of the enzyme nucleic acid and thusidentifying a compound that can be used to treat a disordercharacterized by undesired enzyme nucleic acid expression. The assayscan be performed in cell-based and cell-free systems. Cell-based assaysinclude cells naturally expressing the enzyme nucleic acid orrecombinant cells genetically engineered to express specific nucleicacid sequences.

[0104] The assay for enzyme nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the enzyme proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0105] Thus, modulators of enzyme gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of enzyme mRNA inthe presence of the candidate compound is compared to the level ofexpression of enzyme mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0106] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate enzyme nucleic acid expressionin cells and tissues that express the enzyme. Modulation includes bothup-regulation (i.e. activation or agonization) or down-regulation(suppression or antagonization) or nucleic acid expression.

[0107] Alternatively, a modulator for enzyme nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits theenzyme nucleic acid expression in the cells and tissues that express theprotein.

[0108] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe enzyme gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0109] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in enzyme nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in enzyme genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the enzyme gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the enzyme gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a enzyme protein.

[0110] Individuals carrying mutations in the enzyme gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the enzymeof the present invention. SNPs were identified at 9 different nucleotidepositions, including non-synonymous coding SNPs at positions 2263 and13195 (protein positions 31 and 164, respectively). Changes in the aminoacid sequence caused by these SNPs is indicated in FIG. 3 and canreadily be determined using the universal genetic code and the proteinsequence provided in FIG. 2 as a reference. The gene encoding the novelenzyme of the present invention is located on a genome component thathas been mapped to human chromosome 5 (as indicated in FIG. 3), which issupported by multiple lines of evidence, such as STS and BAC map data.Genomic DNA can be analyzed directly or can be amplified by using PCRprior to analysis. RNA or cDNA can be used in the same way. In someuses, detection of the mutation involves the use of a probe/primer in apolymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

[0111] Alternatively, mutations in a enzyme gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0112] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0113] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant enzyme gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0114] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0115] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the enzyme gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the enzymeof the present invention. SNPs were identified at 9 different nucleotidepositions, including non-synonymous coding SNPs at positions 2263 and13195 (protein positions 31 and 164, respectively). Changes in the aminoacid sequence caused by these SNPs is indicated in FIG. 3 and canreadily be determined using the universal genetic code and the proteinsequence provided in FIG. 2 as a reference.

[0116] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0117] The nucleic acid molecules are thus useful as antisenseconstructs to control enzyme gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of enzyme protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into enzyme protein.

[0118] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of enzyme nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired enzyme nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the enzyme protein, such as substratebinding.

[0119] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in enzyme geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired enzymeprotein to treat the individual.

[0120] The invention also encompasses kits for detecting the presence ofa enzyme nucleic acid in a biological sample. For example, the kit cancomprise reagents such as a labeled or labelable nucleic acid or agentcapable of detecting enzyme nucleic acid in a biological sample; meansfor determining the amount of enzyme nucleic acid in the sample; andmeans for comparing the amount of enzyme nucleic acid in the sample witha standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect enzyme protein mRNA or DNA.

Nucleic Acid Arrays

[0121] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0122] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0123] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0124] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0125] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0126] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0127] Using such arrays, the present invention provides methods toidentify the expression of the enzyme proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the enzymegene of the present invention. FIG. 3 provides information on SNPs thathave been found in the gene encoding the enzyme of the presentinvention. SNPs were identified at 9 different nucleotide positions,including non-synonymous coding SNPs at positions 2263 and 13195(protein positions 31 and 164, respectively). Changes in the amino acidsequence caused by these SNPs is indicated in FIG. 3 and can readily bedetermined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a reference.

[0128] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0129] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0130] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0131] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0132] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified enzyme gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

Vectors/Host Cells

[0133] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0134] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0135] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0136] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0137] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0138] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0139] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0140] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0141] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0142] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0143] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0144] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site maybe introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enteroenzyme. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0145] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0146] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kujan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (invitrogenCorporation, San Diego, Calif.).

[0147] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0148] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0149] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0150] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0151] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0152] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0153] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0154] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0155] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0156] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell- free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0157] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such asenzymes, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0158] Where the peptide is not secreted into the medium, which istypically the case with enzymes, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0159] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

Uses Of Vectors And Host Cells

[0160] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga enzyme protein or peptide that can be further purified to producedesired amounts of enzyme protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0161] Host cells are also useful for conducting cell-based assaysinvolving the enzyme protein or enzyme protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native enzyme protein is useful forassaying compounds that stimulate or inhibit enzyme protein function.

[0162] Host cells are also useful for identifying enzyme protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantenzyme protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native enzyme protein.

[0163] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a enzyme proteinand identifying and evaluating modulators of enzyme protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0164] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the enzyme proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0165] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the enzyme protein to particularcells.

[0166] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0167] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing tansgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0168] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G₀ phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0169] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, enzyme protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo enzymeprotein function, including substrate interaction, the effect ofspecific mutant enzyme proteins on enzyme protein function and substrateinteraction, and the effect of chimeric enzyme proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more enzyme proteinfunctions.

[0170] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 4 1 918 DNA Homo sapiens 1 atgatggacc aaaaaacatg caatgatgca gagcactataatttcaagct ggttgttgaa 60 aactgcatga tctgtgctag tggtagaaaa ggcgaagatgcggggaggct tccaagatgg 120 ccaaatagga acagctccag gctgcagctc ccagcgagatcaatgaggaa gacaagtgat 180 ttctgcattt ccaactcaga tgtctgtgaa aaaattattggaggaaatga agtaactcct 240 cattcaagac cctacatggt cctacttagt cttgacagaaaaaccatctg tgctggggct 300 ttgattgcaa aagactgggt gttgactgca gctcactgtaacttgaacaa aaggtcccag 360 gtcattcttg gggctcactc aataaccagg gaagagccaacaaaacagat aatgcttgtt 420 aagaaagagt ttccctatcc atgctatgac ccagccacacgcgaaggtga ccttaaactt 480 ttacagctga cggaaaaagc aaaaattaac aaatatgtgactatccttca tctacctaaa 540 aagggggatg atgtgaaacc aggaaccatg tgccaagttgcagggtgggg caggactcac 600 aatagtgcat cttggtccga tactctgaga gaagtcaatatcaccatcat agacagaaaa 660 gtctgcaatg atcgaaatca ctataatttt aaccctgtgattggaatgaa tatggtttgt 720 gctggaagcc tccgaggtgg aagagactcg tgcaatggagattctggaag ccctttgttg 780 tgcgagggtg ttttccgagg ggtcacttcc tttggccttgaaaataaatg cggagaccct 840 cgtgggcctg gtgtctatat tcttctctca aagaaacacctcaactggat aattatgact 900 atcaagggag cagtttaa 918 2 305 PRT Homo sapiens2 Met Met Asp Gln Lys Thr Cys Asn Asp Ala Glu His Tyr Asn Phe Lys 1 5 1015 Leu Val Val Glu Asn Cys Met Ile Cys Ala Ser Gly Arg Lys Gly Glu 20 2530 Asp Ala Gly Arg Leu Pro Arg Trp Pro Asn Arg Asn Ser Ser Arg Leu 35 4045 Gln Leu Pro Ala Arg Ser Met Arg Lys Thr Ser Asp Phe Cys Ile Ser 50 5560 Asn Ser Asp Val Cys Glu Lys Ile Ile Gly Gly Asn Glu Val Thr Pro 65 7075 80 His Ser Arg Pro Tyr Met Val Leu Leu Ser Leu Asp Arg Lys Thr Ile 8590 95 Cys Ala Gly Ala Leu Ile Ala Lys Asp Trp Val Leu Thr Ala Ala His100 105 110 Cys Asn Leu Asn Lys Arg Ser Gln Val Ile Leu Gly Ala His SerIle 115 120 125 Thr Arg Glu Glu Pro Thr Lys Gln Ile Met Leu Val Lys LysGlu Phe 130 135 140 Pro Tyr Pro Cys Tyr Asp Pro Ala Thr Arg Glu Gly AspLeu Lys Leu 145 150 155 160 Leu Gln Leu Thr Glu Lys Ala Lys Ile Asn LysTyr Val Thr Ile Leu 165 170 175 His Leu Pro Lys Lys Gly Asp Asp Val LysPro Gly Thr Met Cys Gln 180 185 190 Val Ala Gly Trp Gly Arg Thr His AsnSer Ala Ser Trp Ser Asp Thr 195 200 205 Leu Arg Glu Val Asn Ile Thr IleIle Asp Arg Lys Val Cys Asn Asp 210 215 220 Arg Asn His Tyr Asn Phe AsnPro Val Ile Gly Met Asn Met Val Cys 225 230 235 240 Ala Gly Ser Leu ArgGly Gly Arg Asp Ser Cys Asn Gly Asp Ser Gly 245 250 255 Ser Pro Leu LeuCys Glu Gly Val Phe Arg Gly Val Thr Ser Phe Gly 260 265 270 Leu Glu AsnLys Cys Gly Asp Pro Arg Gly Pro Gly Val Tyr Ile Leu 275 280 285 Leu SerLys Lys His Leu Asn Trp Ile Ile Met Thr Ile Lys Gly Ala 290 295 300 Val305 3 17321 DNA Homo sapiens misc_feature (1)...(17321) n = A,T,C or G 3ttaaaaatta gccaggcatg gtggtgcatg cctttagtcc cagcttctca tgaggctgag 60gtaggacgat tgctggagcc caggagttgg aggctgcagt gagctgtgat ggttccactg 120tactgcatcc tggatgagct gtgatcacac cataactctt cagcctagat gacagagtga 180ggccctaatt caaaaaaaga aaaaagagag acagagagaa agcccagggg ttggcccact 240cccatcattg ggaataaaga aaggccttta gggttctgtg cacttctggc aaaacctcca 300tactccttgc cccccaccac ttacagcctg attccaacag ggcatcctag gccctgaagg 360acactccctt ccctgcccct cagagggcac tccttggccc actcccctgc tacattttcc 420tccatggcat ctaacacacc ctgatatact ttagatggtg tatgtctgcc tgcctccctc 480actggaacct aagctccaca taagagatac ttttgtctac ttacttctat gacctccaat 540acctagaaca acagtatgtg atacataatt aggccctcga taaatatttc ttgaataatt 600ttgaataaga gatggaatta agccagacag tggcaaatgt aatcaaaagc agagaaggga 660ttctagactc tggtttcaag caagtggtag aagtcaggtc tccttcagga cctgaaaatc 720ttacctttag aaaggaatga ggtgggtaca ggaggtgagg ggcccatgct gttagagctg 780ggaaactcct tcccacctat gacactatgg ccaatagtgt gatgctgcga gggaatccac 840aaagaaggtg tcaagacttg accagggata gttaactctc cagggctaaa acctaattgt 900gcggccatgg tagtcctatg cagttgacca taagagagct tgaatgggga ctgctcctta 960caaacatgtg cttcccagac tctgatacac tcattaagcc ccaaagtccc ttttgaagtc 1020tccctcctcc ttgtcctcag atagcaagca tgcaggctgt gaggaagcat gagtgggtga 1080gaagtcggaa agttgcacat gcacatttca agtcctgttc tatagaaagg ctctcatctg 1140tggccatttc acctcacaat gttagcaagg tggggggcag gagcttcagt aatcagaagg 1200agcaccgagg aagtggggac actctaatgt caggaagcat cgcctgaaag caatgcaatc 1260ataggaatgt gaccattgct gtggtgccgt tgtgttttta gcagttccac aataccagaa 1320gcccctgcaa tttcaaactc agcttaaggc aagaaaaaat aaaagaacta gaatcacctc 1380cctctaccct ctactcaacc tgaatcatac tccatactct ccctgccttg actttgtgga 1440aaaatagaaa tcaaggtggc gagcaaagaa gaggacttag ggaatgaaag aaaaggcaaa 1500gtataggtga gcaaccgaaa gcaggtgaaa cctgcaggct ggtttgctca tgctcaaaag 1560tagggaagac acatttctac caaaatggaa aatgaggaca ctggtccctt tcccaccctg 1620ccccactgca ccaggatccc gagttttctt ccactggtca cagtaccagt cagttcacga 1680atctaagact gcctgaataa aaacgtttgg attctcagca caggttttca accacagaac 1740tgtcagccac caccatataa ttttatcact gacaggtcaa ctgaactgaa gcttacttag 1800actagcctag caaagcactt ctgaaaaaag tgtcctgtaa ctttttaaaa atcaattacc 1860gattgtcaat acaggttggc acatgtctta agccatgtcc ctcataagac tacagaagca 1920agtatatatt tcaatcaggg ctattttgtt gtttttgtta ctgtcatacc tgcccaaaac 1980tccatttcct gtctgttttc tagctggaag ttaaagcaac catgagcaaa gctgtaggaa 2040acttcatcta ccaaaaacag gacaggatgt aaaacttcac accgagtgtc atgtggcagg 2100atggggaagc acaaaaagag actcatgcaa agttttgaaa acaccttgag agaagtgaac 2160aaacatcact atgatggacc aaaaaacatg caatgatgca gagcactata atttcaagct 2220ggttgttgaa aactgcatga tctgtgctag tggtagaaaa ggcgaagatg cggggaggct 2280tccaagatgg ccaaatagga acagctccag gctgcagctc ccagcgagat caatgaggaa 2340gacaagtgat ttctgcattt ccaactcagg taccttgttc atctcattgg gactggttgg 2400acagggtggg cagcccagga agggtcagac gaagcagggc agggtatcgc ctcactgggg 2460aagtgcaagg ggtcagggga tttccctttc ctagccaaag gaagccatga cagtctgtac 2520ctggaggaat ggtacactcc tgcccaaata ctgcactttt cccatggcct tcgcaaccag 2580tagaccagaa aattccctcc catacctggc tcggtgggtc ccacacccac ggagccttgc 2640tcactgctag agcagcagtc tgagatcaat cttcaatgct acagcttggc agggtgaggg 2700atgtctgcca ttgctgaggc tggagtaggt ggttttgtgc tcacagtgta aacaaagagg 2760ccaggaaact cgaactgggt ggagcccact gtagctcagc aaggcctact acctctctag 2820attccacttc tgtgggcagg gcatatcaga acaaaaggca gcagacagct tcagcagacc 2880taaatgtccc tgtctgacag ctctaaagag agcagtagtt ctctcagcat gacgttcgag 2940ctctgagaac ggacagactg cttcctcaag tgggtccctg acacccatgt agcctgactg 3000ggagacactt cccagtaggg gctgacaaac acctcataca ggcagggggc ccctctagga 3060tgaagcttcc agaggaagga tcaggcagca atatttgctg ttctgcagcc tccactagtg 3120atacccaggc aaacagggtc tggagtggac ctccagcaaa ctccaacaga cctgcagctg 3180agggcccaac agttagaacg aaaactaaca aacagaagaa atagcatcaa catcaacaaa 3240aagcacatcc ataccaaaac cccatctgta ggccaccaac atcaaaggcc aaaggtaaat 3300aaaaccacaa agatggggag aaaccagaga gcctcttctc ctccaaagga tcgcagttcc 3360tcaccagcaa cagagtaaaa ctggacagag agtgagtttg acaagttgac agaagtaggc 3420atgagaaggt cagtaatagc aaacttctct gggctaaaga agcatgttct aacccattgc 3480aaggaagcta aaaaccttga aaaaagatta gacgaatggc taactagaat aaatagtgta 3540gagaagatct taaatgacct gaaggagctg aaaaccatac cacgagaact tcgtgacacg 3600tgcacaagct tcaatagcca attccatcaa gtggaagaaa ggatgtcagt gattgaagat 3660caaattaatg aaatcaagca agaagacaag attagagaaa aaagagtgaa aagaaatgaa 3720taaagcctcc aagaaatatg ggactatgtg aaaagaccat tttgatttgt gttcctgaaa 3780gtgatgggga gaatggatcc aagttaggaa accttcagga tattatccag gagaatttct 3840ccaacctagc aaggtaggcc aacattcaaa ttcaggaaat acagaaaaca ccacaaagat 3900actccttgag aagagcaaac ccaagataca taattgtcag attcaccaag gttgaaatga 3960agggaaaaat gttaagggca gccagataga aaggtcaggt tacccacaaa gggaagccca 4020tcagactaac agcagatctc tcagcagaaa gtctacaagc cagaagagag tgggggccaa 4080tattcaacat tcttaaagaa aagaattttc aacccagaat ttcatatcca gccaaactaa 4140gcttcacaag tgaaggagaa ataaaatcct ttacagaaaa gcaaatactg agaaatttca 4200tcatcaccag gcctgtctta caagagctcc tgaaggaagt actaaacatg gaaaggaaca 4260actggtacca gccactgcaa aaccatgcca aattgtaaag accatcaatg ctaggaagaa 4320actgcatcaa ctaacgagca aaataaccag ctagcatcat aatgacagga ccaaattcac 4380acataacaat attaatctta agtgtaaatg ggctaaatgc cccaattaaa agacacagac 4440tggcaaatgg gataaagagt caagaccaat cagtgtgctg tattcaggag accaatctcc 4500atgcaaagac acacataggc tcaaaataaa gggatggagg aagatctacc aagcaaatgg 4560aaagcaaaaa aagcaagggt tgcaatccta gtctctgata aaacagactt taaaccaaca 4620aagaccaaaa aagacaaggc cattacataa tggtaaaggg atcaatgcaa caaggagagc 4680taacaatcct aaatatatat gcacacaatt caggaggacc cagattcata aagcaagtcc 4740ttgaagacct acaaagagat ttagactccc acacaataat aatggaagac tttaacaccc 4800cactatcaat attagacaga tcaacgagac agaaggttaa caaggatatc caggacttga 4860actcagttct gcaccaagca gatctaatag acatctacaa aactctccat cacaaatcaa 4920tagaatatac attcttctca gcaacacatc gcacttaatc taaaattgac cacataattg 4980gaagtaaagc actcctcagc aaatataaaa gaacagaaat cacaacaaac tgtctctcag 5040accacagtgc aaccaaatta gaactcagga ttaagaaact cactgaaaac cacacaacta 5100catgggaaac tgaacaacct gctcctgaat gactactggg tnnnnnnnnn nnnnnnnnnn 5160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnctatttat 5880gacaaaccca cagccatatc atactgaatg gacagaaact ggaagcattc cctttgaaaa 5940ctggcacaag acaaggtatg ccctctttca ccactcttat tcaacatagt gttggaagtt 6000ctggctacag caatcaggca agaaacagaa ataaagtgta ttcaactagg aaaacaggga 6060gtcaaattgt ctctgtttgc agatgacatg attgtatatc tagaaaaccc catcatctca 6120gcccaaaatc tccttaagct gatgagcaac ttcagcaaag tctcaggata caaaatcaat 6180gtgcaaaaat cacaagcatt cctatacacc aataacagac agagagccaa atcatgagtg 6240aactcccatt cacaattgct tcaaagagaa taaaatacct aggaatccaa cttacaaggg 6300atgtgaagga cctcttcaat gagagctaca aaccactgct caacgaaata aaagaagaca 6360caaacaaatg gaaaaacatt ccatgctcat ggataggaag aatcaatatc atgacaatgg 6420ccatactgcc caaagttatt tatggattca atgccatccc catcaagctt ccaatgactt 6480tcttcacaga attggaaaaa actactttaa agttcatatg gaaccaaaca agagcccaca 6540ttgccaagac aatcctaagc caaaagaaca aagctggaag catcatgcta cctgacttca 6600aactatacta caaggctaca gtaaccaaaa cagcatggta ctggtaatgg ctgcacaact 6660atgcaaatat actaaaatcc attgactcat acactctaaa tgggtgacct ttatggtgtg 6720tgaattatgt ctcataaagt tgttagaagt caacataaat ggaagagcaa ccattcacat 6780aaaaataaac aaaattgtca atgttttaag aatttttcag taggtgtagt taattacaat 6840ttgacttttt taagtctgca ctaaaattac tcaccaaaac caatagcagg gtcctcactg 6900ctgttactga aaatgattaa cctttgatac acttgtaata tctgagaaaa agaaatgcag 6960gggtctcagc agggctccct tctaaggtca cttgatttct aaagaagtaa ccactaggtt 7020tgaagtcatc aggatgttaa ctatggggat ggttggttca gtacccaaca tcctgacagc 7080acatctgacc atgtatattg tatcggagac cacatcctca gctcagaaaa agagctgaac 7140tcatttcaaa tagaaagcac actgcatact gttcctccag ggactgaggt tgactcttct 7200tagagtgaga cattccccaa cattggaaca aaaatgactc ccacttcttt tctcacctaa 7260acctgttcag aagaaagtaa ggaagaaagg caggaagcag gggtcggggg gggcggggag 7320ggagggaaac tcggagatac tttcagtatc taaagttgtg aaactagaca atcaggaacg 7380cacaatcaga gggctgaaaa gggccaagag cccctaccct cctccagccc atgttcccac 7440acctgccaca gaccaggcag gagcacaata aacactcacc acaaagtggg gaaggaaaat 7500ccaggcagga gcctctatgt aaataaatct ccctcctgtc ctcagcttgc acttggccta 7560ctaattctta tataatccaa ggagacagct agaaagaatt ttgattggtg accaattttg 7620aggactttta ttaaaattct aatttaagtc ttcgagagtt tccagtcatg gatactacag 7680ataatattgc agatgatgaa agcgtcttca aaatcatagc tgagacactt tacagtcttc 7740cctggtgtac tctgatggca acaaggtccc ttgcccctct cctaccctgt agaattccag 7800cgcccccctc agcagtccta gcaaaggaaa gcctgcctgc tggcagtgag ccatcatcca 7860ccattctcac ttatttggat ttggtttcct aatgctaaac tttgaaactt gaaaaaaaaa 7920atgaaaggaa accacatcct ttttactctc agtatataga tagaggcagt taagaactga 7980aaacagattt tcaggttgat tgatgtggga cagcagccac aatgaggaac tcctatagat 8040ttctggcatc ctctctctca gttgtcgttt ctctcctgct aattcctgaa ggtaagactg 8100attcttctca tttccattat gaccatgaag caaaatggag cagactttca atgaactttt 8160ggcaatatta ccttcctata ctgaaagaag atgttttccc ccaagcctta taatgtttag 8220ccaggcacaa tggcacatgc ctgtagtccc agctactagg aacgctgagg tggaaggatc 8280acttgagcgc aggagtttga gtccaggctg ggcgacatag tgagaccctg tctctatttt 8340ttttttttaa atagcattct ttcttggatt ttaaatatgt ctgtatttta gaaaaaaaaa 8400atcttttctt ctcataatta ttttgcaaaa gatagcagaa ccaccaatgc ttattgctgc 8460caatttgtaa cttgaaaatt gccaaaaagg acaatgtaaa aatgagttaa caaaactctc 8520ttgttcgtaa aacaaatagt accttccagg aaaggaatag tggcaactta gtcagctcag 8580cctttcctgc tgcagaaaat gcatagataa agacaattaa gacaaatctg acctagacac 8640caaaagtgaa tcaggaatta ttagaatttt tatacttatt tctctggtct agagactctt 8700tggcaataaa ttaaggcgtg aggccagttc cctcattaac cctttgggtg tgctatagta 8760ttttttatta agctaatttt ttgtctacca aatggtaacc aatatttctg cttttgtgtg 8820agttgagcat gcatgggtta aaatgctgta taattcatgg tattaccact gaaagggtga 8880cagctcttgg ggagctctag atttcccttg aagaacctgc taggaagtga ggactactct 8940caagctgtgg caatatggag ccagccacgg tttttcccag gattcatcag cttttggagg 9000cagggactta tgtaataacc tgccttgcca ggaattccca aagaactctg gtttatcata 9060tttaggtgca tatctggttt atgcacctaa atatgagaag actttgggag gctgaggcgg 9120gtgtttcaca aggtcaagag atggagacca tcttggccaa catggtgaaa ccttgtctcc 9180actaaaaaaa caaaaattag ctgggtatgg tggcacgtgc ctgtaatccc agctacgcag 9240gaggctgagg caggagaatc gcttgaacca gagagttgga ggctgcagtg agctaagatc 9300gtgccactgc actccagcct ggtgacagag cgagactctg tctcaaaaaa aaaacaaaaa 9360agagagagag agagaagaaa tataaaaata acaaggctgc aggacatttg ggacaaaggt 9420tggctatgtt ataagcaggg catagtatga ctatattcag gcatgttaaa tcctccataa 9480cagaaaaaca cagggcagat aaatccagcc atatcttttc ttcaaaggaa aacaacaaaa 9540gcaacctgct gtgctcctct ttgtttcctg tgttcagaga gggcagtgtg ggcccatata 9600aggaacagac agcaaagcaa ggcacagttg aaatctatct gtacatgaat caccctagga 9660aaattgggat tgggtgaagg tcttttgttt ctataagctc tttacccttt tgtttctaga 9720gcctagaatt tcttcctccc agtttccttt ttaaaagcag agacctctgc tttttcaaat 9780cacactagcc acatttcatg tggtcactct cctccttgct cttaccctca tcaaatctta 9840cgaaaggctt ctatagctgt cattaatact tgcctgtgaa tttggcaatg acttagtcct 9900aattatggaa caagaggcac tgtgccctat tcaagaaacc atgagtctca gcaaaactat 9960tttgggagaa tccaagaaca tctggtgcag gaggtggagc actcggagtg tatcactaag 10020acataaggtt ctctctatct gcatgtatgg tttgccttgt gtttagttct cactgttcct 10080taacaatggg tctctacttc aagactatca tcaaggaaat gtcacttagg cagaattctt 10140ggcatatggt atcttctctc ttctcttccc tatctcattg gaaaagacct tagattaaac 10200cccatggttt ggtctagaga ttgaagggga ggtgcaatcg atgttatttt cttaacttat 10260gagtgagaac agtgcagttt aataaccaaa agtctcataa tgaccaaagg actgaggggt 10320agggtcacag ctaaacgatg ctaatatcgg ttcataagct atatttttgc aatggcttgg 10380ggttaaaagg gaagttatga ggcttctaaa agtcttatct atttccccca aattaatagt 10440tagccaaaaa gggtgggagc acttgaggtt gtcactacca ctggcatttc ctgcagcaac 10500tgttgatggt aaagaccaag gcagagcttt ctgccctggg gcctgaggtt gctggtttgt 10560gacatccagc ctaatgatcc tggaacagtc aatggcctcg cttctgtggg tggaaaaaca 10620ggaccctaga gtccaacagg aataggattt gcatcagatg tctgcgggtc tacatagagt 10680aaaagcaggg gaaatgctct catccagact tcctctctga gtgcaaaaag catggctggt 10740cttagaaaca tgactttgtg tagagctctt tgggggtgag caccaatctg atttgtcttt 10800ttccattgaa cagatgtctg tgaaaaaatt attggaggaa atgaagtaac tcctcattca 10860agaccctaca tggtcctact tagtcttgac agaaaaacca tctgtgctgg ggctttgatt 10920gcaaaagact gggtgttgac tgcagctcac tgtaacttgt aagtgcctgg gtttttaaaa 10980aaaagtttgt aaagatactc taatttgggg aaacattaat aaaaagagta ggccgggcac 11040agtggcttac acctgtaatc ccagcacttt gggaggccaa ggcaggtgga tcacatgagg 11100tcaggagttt gagactagcc tggccaacat ggtgaaaccc catctctatt aaaaatacaa 11160aattagccag gcgtggtggc gcatgcctgt aatcccagct actttggagg ctgaggcagg 11220agaattgttt gaacctagga ggcagaggtt gcagtgagcc gagatcgcac cattgcactc 11280cagcctgggc aacgagagtg agactccatc tcaaaataaa ataaaataaa ataaaataaa 11340ataaaataaa ataaaaaata aaacaaaata aaataaaata aaatataaaa taaaataaaa 11400taaataaaat aaaataaagt agatcccact gaagccatgg gaggttcata tgtatcttca 11460catcgcttaa gtctttgggc acttgcttga gcctaaaagg aagttaggcc cacacttgcc 11520acaaagaatc aggcaatgag gaagcagcat ccacaagctc tttctgtaca tctccactga 11580gccattgggt acaggtgctg cagagctttg cttctgtcat gactgcgtga ccaggtagaa 11640ctagcctcag catggaaggg tgaaactttc agtgccccct cagagccctg ggtgcaacca 11700tcaagtacag aaagcttgtg tccaatgacc ttcttgtatg cctccgtgaa ttcctctcac 11760atctttggcc atctcttcac tcctgtcgac cactacattc aaacctcttc ccaggcctgg 11820tcttttccta ctctctaaat caagcctcta aacctaggcc attcaatatg ataggcacta 11880gctacatgta gctatgtaaa tttcaatcaa ttaaagttaa ataaaatctc aaattagttc 11940ctcagttgca ctagccacat ttcaagtcct cagtagccac atgtggctag tgcagattca 12000tagaacattt catcataaaa atatgctcta ctggaaagca ctgttctaag gtctttttct 12060tttatttttt aattatttac tttttattga catataatag ttgcacatat gttggggata 12120catgtgatca tttaagtgag actaatctca gctgatgtaa tggaaaagag gcagtgtgca 12180taatctggtt ggtgtagttt atactaggtt atgaatgata gccttaatcc aatcatcagt 12240acatcagaaa gcattttgat tttgataacc aaaatgaacc tttaagagaa aaaaagactc 12300ctggacccct ccttcagacc accatccctt caacacactc aatgccaagc acactcaatg 12360ccaagcaaat agggtcctcc atggggcaat ctgtttctga aagttttccc atttgagttt 12420gttgttttct tgaaaagatc ccttcagtgc attactttac aactattggc tgctttttgc 12480ttctgtctct ggactactga gatctttgct tcccatcaca gctaataagt aattctccat 12540cacagctaaa taaggcaggg gtagggggca gataagcaca agacttggct ttgctgccat 12600ggaattctct tgtttttcaa tgaacctagt ttagcttttt ataaataagc tggtgatgat 12660gatgatgacg ataaattcat aaacattctt ttattcataa acattctttt attctttaaa 12720gcacacattt tctttctgag ccacaaagaa atctccccac ctccatattt acaaatggga 12780caatgaaact gatcagtata tatgctcaac tgccaataca aagaataaat ccagtaaata 12840atgttgtgtc tgttctcagg aacaaaaggt cccaggtcat tcttggggct cactcaataa 12900ccagggaaga gccaacaaaa cagataatgc ttgttaagaa agagtttccc tatccatgct 12960atgacccagc cacacgcgaa ggtgacctta aacttttaca ggtacgtatc tttgattgtc 13020ttctcaaaag tcatagaacc ttctacaatc tggccgccga cgtgaaaacc tttccttggt 13080gcccctgttg taaaaactca caataaagag gatcctgaaa ttttggacta taatttaaaa 13140tgtaaatatt tatcttatcc cattaacact ttatgttttt cttccaacag ctgacggaaa 13200aagcaaaaat taacaaatat gtgactatcc ttcatctacc taaaaagggg gatgatgtga 13260aaccaggaac catgtgccaa gttgcagggt ggggcaggac tcacaatagt gcatcttggt 13320ccgatactct gagagaagtc aatatcacca tcatagacag aaaagtctgc aatgatcgaa 13380atcactataa ttttaaccct gtgattggaa tgaatatggt ttgtgctgga agcctccgag 13440gtggaagaga ctcgtgcaat gtaagtaaaa taagatccca cgtttcagct attgaattaa 13500gtcatgcaga cagagaaaat gtaatgtcat gctttgtttt gtcatggcac tggcttctac 13560aggatcctag agctttttga aataagttta ataaaacccc agtcaaataa atgaatgctc 13620gcagctcaag tgagtcatta atcaaaaata agaagttcac tgctagtgca tggggtgacc 13680tactacatat ttttgatttt ttacaaaaat tactaagaag caatgtttgg tctcattttt 13740catgttaaca tacgtagatc taaacttgaa gatatacaac acttatgaga gctgaagtca 13800gggttcctca aggccactca gtactctttc cccatttcag tacattccct ctccttcccc 13860actacgttcc tctgccattt aacatcggca tgtctgaaat gtctgtcaag cctggctgaa 13920tatttccagc cagaccccat agcttcctag agcttagttt aatctgcagc aaacttcaaa 13980gcatgactca taccagaaaa tgtgtaagct tccatcctag aaccccctga gaagaggtgg 14040gatagggcca tgcttaaaga tgagaataga agaaacattt tgcagatttt taaactgagt 14100actaccttgc atttctcatt tgtcccaaag accctataga gccttacctg aagaaaggtg 14160tgcagtatgc atggtttggc agttaccaga gactgagaaa tccaaaggtc tctgtccttc 14220cctagatcta tgtcttgctt tcagaaccaa agctagccaa atcccaggaa aagaaaacat 14280ttgtcttcag tacctgtcca ctttctcttc tctgccctag ttcccccatt ccctcccttg 14340tccaccaccc tgggttccca ggaacccaga accttcatgc ttttggaacc cacatgcagc 14400atcctaaggc cacggcagca tcccaagacg ctagtcccca tcccaccagc cacccgccac 14460caccgaaact tggcaatcct tgcctgttct ctcaacaccc tggaattggc tattgatgaa 14520ttaaacctaa aggactaatt ctgcaaatgc cagttactgt aacaggcagg ggaaataaac 14580ctgttcaggg aggatcccgg gttccaacat ggttctttat ttcatatctt tataaggcca 14640acttttgcct gaaagggact gatttggttt tgtttctttt ggaaggcaat tatctgctag 14700aagaaccaga aaacatagtg tttattcttt gcttcaatgt atcatctgca tttgactatt 14760ttgccccttg agttattaag cattttgaga aaacgacaaa taaacaggag actttccttt 14820ccaaaagcag agcaatagtc tcaaaattag ctgataacat gtacaagttt cacttcgatt 14880ttcactggct cataaataaa ccaagtgaac caattcaaaa atattaaaat atttccaaac 14940attttaattt ttaaaattaa agcactatct tcaattaagt caaggttggt cttaactgca 15000tattaaatgc tgcagaattt cttccatttc actagtggta atgctgaaca ctgaccccac 15060accctacccc tcttgttttc ctccagggag attctggaag ccctttgttg tgcgagggtg 15120ttttccgagg ggtcacttcc tttggccttg aaaataaatg cggagaccct cgtgggcctg 15180gtgtctatat tcttctctca aagaaacacc tcaactggat aattatgact atcaagggag 15240cagtttaaat aaccgtttcc tttcatttac tgtggcttct taatcttttc acaaataaaa 15300tcaatttgca tgactgtacc tgtttctctc ttgtaacctt agtgggcaga tctgcgccag 15360caaagtgaag cagagttaca tggcagcctt gtagataatg caaggattac gtgatcaatg 15420tcaccagagt catttgtgcg tcaagtgaca tgggaatgct tcctgaatta ttcatctctc 15480tgtctccatg aaatccagca agaacaaccc taagatcagg tctaataaca aatgcagtaa 15540aggcctccac aattccacag gaaacagaaa cagccagtct ggaacatttg tgttaaaagg 15600aaatataggg cattgttcca cattaacacc tgcttttcat gttatacaca agaacctgag 15660tctatgggag aaaaagaaac aagcaatgcc ttacggtatt ttccaaattc tagggcatac 15720aaataaagtg cttgggcagc caagaaaatt atacaataga gagtcagcct tctttctcag 15780tgccttgcat ttaagccttg gattacctag gtgatttctc agttctcttt gttcttgaga 15840cggggtagat ccaatcaaca tggtggttac agtggctgcc atcccatagt agggagtgag 15900tgttgacttg tagccagaaa gttttctttg gaaaaaaccg tcataatatt ttttcaacat 15960agtgaattta gtaggtagtc atcagcctgc agtcatttca agatgtgatt atgtgaacag 16020attctttcca tttctatagt ccctttagta atataaattt tatatatgta tattatatat 16080tatgtaatac aaatttcaat atatacatat attatatatg tatatacaca tacacacata 16140tatatacaca tatacacaca catatataca tggataatac atattcaaac caataacata 16200cagcaacaca aagagacagg ctcttaaaat aaagtgcaaa aaaaaaaaaa aaccaacaac 16260aaaggaagga gcatagctca aagactggct aaaataatag taaattcgtt ttcaaccatg 16320gtagttcatt agactcacca agctctttaa aattaccaat gcccaggtcc cacctctaga 16380gattctgatt taattggtat ttggtgggac ccaggatgta gtattctttg tatgagctct 16440ccagtgattc taatatgcag ccagggataa gaacccttat actagaaaag gagatctggt 16500gctactatat aaaagagagg ctctgagaga gtaactacga tgatatttta aatacttgct 16560acaatccctc attgctagtt aggattttaa ctgcaaaaag aggatggaat attccctact 16620gtcaatatgt attacagatt ttgaactctg tttaaaaaat ggttgtatgg aacccttgtt 16680aattgctggt agaagtgtaa attagtacaa cttcatagga aaattggcag tgtctactaa 16740aactcaacat atatataaac ctatgaccta gcaattccat tcctgggtag atcccaaaca 16800cacatgagtg cttttatcca ccaaagatgt gttctggaat gtacacagca gctttattct 16860tggaagtcaa aactagaaat aactcaaata tccacacaca ataaaatgga taaacaacgt 16920tacacaggca tacaataaaa tattacacag caatgaaaaa gaacaaactt cagttttatg 16980caacacaaat ttaatgttga ggaaaagtga gataaaaaag aatatgcatg atgagattcc 17040atttatctga aagttggcat aaggaaaaac taatccatgg ggatagcagt cagaaaaatg 17100tagagctgct aactaggagg gggtacgagg gatcccttta gggtactaga gatgttcgta 17160tcttgatcta gatgttggtt acacacttat ttattcataa atttaaaact catcaagcta 17220taaagactta tgtgctttac tatatgtgaa ttattattct gtgaaaaaat tttaattaca 17280aaatttaaaa ataaattgta gcccaggctt ttgtgtgcta t 17321 4 261 PRT Homosapiens 4 Arg Asn Ser Tyr Arg Phe Leu Ala Ser Ser Leu Ser Val Val ValSer 1 5 10 15 Leu Leu Leu Ile Pro Glu Asp Val Cys Glu Lys Ile Ile GlyGly Asn 20 25 30 Glu Val Thr Pro His Ser Arg Pro Tyr Met Val Leu Leu SerLeu Asp 35 40 45 Arg Lys Thr Ile Cys Ala Gly Ala Leu Ile Ala Lys Asp TrpVal Leu 50 55 60 Thr Ala Ala His Cys Asn Leu Asn Lys Arg Ser Gln Val IleLeu Gly 65 70 75 80 Ala His Ser Ile Thr Arg Glu Glu Pro Thr Lys Gln IleMet Leu Val 85 90 95 Lys Lys Glu Phe Pro Tyr Pro Cys Tyr Asp Pro Ala ThrArg Glu Gly 100 105 110 Asp Leu Lys Leu Leu Gln Leu Thr Glu Lys Ala LysIle Asn Lys Tyr 115 120 125 Val Thr Ile Leu His Leu Pro Lys Lys Gly AspAsp Val Lys Pro Gly 130 135 140 Thr Met Cys Gln Val Ala Gly Trp Gly ArgThr His Asn Ser Ala Ser 145 150 155 160 Trp Ser Asp Thr Leu Arg Glu ValAsn Ile Thr Ile Ile Asp Arg Lys 165 170 175 Val Cys Asn Asp Arg Asn HisTyr Asn Phe Asn Pro Val Ile Gly Met 180 185 190 Asn Met Val Cys Ala GlySer Leu Arg Gly Gly Arg Asp Ser Cys Asn 195 200 205 Gly Asp Ser Gly SerPro Leu Leu Cys Glu Gly Val Phe Arg Gly Val 210 215 220 Thr Ser Phe GlyLeu Glu Asn Lys Cys Gly Asp Pro Arg Gly Pro Gly 225 230 235 240 Val TyrIle Leu Leu Ser Lys Lys His Leu Asn Trp Ile Ile Met Thr 245 250 255 IleLys Gly Ala Val 260

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 2. An isolated peptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 3. An isolated antibody that selectively bindsto a peptide of claim
 2. 4. An isolated nucleic acid molecule consistingof a nucleotide sequence selected from the group consisting of: (a) anucleotide sequence that encodes an amino acid sequence shown in SEQ IDNO:2; (b) a nucleotide sequence that encodes of an allelic variant of anamino acid sequence shown in SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotidesequence that encodes an ortholog of an amino acid sequence shown in SEQID NO:2, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment ofan amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids; and (e) a nucleotidesequence that is the complement of a nucleotide sequence of (a)-(d). 5.An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence thatencodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids; and (e) a nucleotide sequence that is thecomplement of a nucleotide sequence of (a)-(d).
 6. A gene chipcomprising a nucleic acid molecule of claim
 5. 7. A transgenic non-humananimal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acidvector comprising a nucleic acid molecule of claim
 5. 9. A host cellcontaining the vector of claim
 8. 10. A method for producing any of thepeptides of claim 1 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 11. A method for producing anyof the peptides of claim 2 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 12. A method for detecting thepresence of any of the peptides of claim 2 in a sample, said methodcomprising contacting said sample with a detection agent thatspecifically allows detection of the presence of the peptide in thesample and then detecting the presence of the peptide.
 13. A method fordetecting the presence of a nucleic acid molecule of claim 5 in asample, said method comprising contacting the sample with anoligonucleotide that hybridizes to said nucleic acid molecule understringent conditions and determining whether the oligonucleotide bindsto said nucleic acid molecule in the sample.
 14. A method foridentifying a modulator of a peptide of claim 2, said method comprisingcontacting said peptide with an agent and determining if said agent hasmodulated the function or activity of said peptide.
 15. The method ofclaim 14, wherein said agent is administered to a host cell comprisingan expression vector that expresses said peptide.
 16. A method foridentifying an agent that binds to any of the peptides of claim 2, saidmethod comprising contacting the peptide with an agent and assaying thecontacted mixture to determine whether a complex is formed with theagent bound to the peptide.
 17. A pharmaceutical composition comprisingan agent identified by the method of claim 16 and a pharmaceuticallyacceptable carrier therefor.
 18. A method for treating a disease orcondition mediated by a human enzyme protein, said method comprisingadministering to a patient a pharmaceutically effective amount of anagent identified by the method of claim
 16. 19. A method for identifyinga modulator of the expression of a peptide of claim 2, said methodcomprising contacting a cell expressing said peptide with an agent, anddetermining if said agent has modulated the expression of said peptide.20. An isolated human enzyme peptide having an amino acid sequence thatshares at least 70% homology with an amino acid sequence shown in SEQ IDNO:2.
 21. A peptide according to claim 20 that shares at least 90percent homology with an amino acid sequence shown in SEQ ID NO:2. 22.An isolated nucleic acid molecule encoding a human enzyme peptide, saidnucleic acid molecule sharing at least 80 percent homology with anucleic acid molecule shown in SEQ ID NOS:1 or
 3. 23. A nucleic acidmolecule according to claim 22 that shares at least 90 percent homologywith a nucleic acid molecule shown in SEQ ID NOS:1 or 3.